| Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst)is one of the most devastating diseases in wheat worldwide.Breeding resistant cultivars is an effective and environmentally friendly measure for the control of wheat stripe rust.However,the rapid variation in virulence of Pst usually leads to a loss of wheat resistance,which results in frequent disease epidemics and causes huge production loss.Therefore,analysis of the pathogenic mechanism of Pst and wheat from the molecular level is of great significance for disease control.As an obligate biotrophic fungus,Pst forms a specialized structure called haustoria during infection,which participates in the exchange and transmission of nutrients and messages between host and pathogen.Haustoria can secret effectors to manipulate host innate immunity,thus facilitating its infection.Based on the sequencing of Pst genome in the previous study,we obtained two effectors named Pst＿9302 and Pst＿148 through bioinformatics analysis and large-scale functional screening.Both effectors can inhibit Bax-induced cell necrosis,aimed to reveal the mechanism of host immunity inhibition by two effectors.Further study proved that they can also inhibit host PTI(PAMPs-triggered immunity)and ETI(Effectors-triggered immunity)responses.Therefore,in this study,we further identified the interaction targets of effector Pst＿9302 and Pst＿148 in host cells based on previous research,and analyzed the function of the targets.The main results are as follows:(1)Through screening the cDNA library of the compatible interaction between wheat and Pst by Y2 H,50 and 60 candidate targets were obtained for Pst＿9302 and Pst＿148,The results showed that 9-6Y(Voltage-dependent anion-selective channel protein)and9-9Y(the Elicitor-responsive protein 1)could interact with Pst＿9302,and 15Y(transcription factor ASG4-like)and 46Y(the protein disulfide-isomerase LQY1)could interact with Pst＿148.Bi FC(Bimolecular Fluorescence Complementation)assays revealed that Pst＿9302interacted with 9-6Y and 9-9Y in cytoplasm,and Pst＿148 interacted with 15 Y in nucleus,while the interaction between Pst＿148 and 46 Y in both cytoplasm and nucleus.Interactions of Pst＿9302 with 9-6Y,Pst＿148 with 46 Y were further verified by Pull-down assay.Meanwhile,Pst＿9302 was found to interact with itself in Y2 H assay.Mutation of the four cysteine residues of the Pst＿9302 to serine individually or simultaneously did not affect the interaction.Meanwhile mutation of the FKC and FQC domain of Pst＿9302 to AAA,either did not affect interaction.Further,gel filtration chromatography analysis revealed that Pst＿9302 was able to form a trimer.(2)To further analyze the function of the interaction targets,q RT-PCR was performed to evaluate the experiment pattern during wheat-Pst interaction.The results showed that the9-6Y and 46 Y were both significantly up-regulated in the early stage of wheat and Pst compatible interaction.9-6Y was highly expressed at 12 hpi and 24 hpi after infection by virulent Pst,while 46 Y was highly expressed at 24 hpi.Using the virus induced gene silencing(VIGS),9-6Y or 46 Y was silenced in compatible and incompatible interaction between wheat and Pst.The silencing efficiency of 9-6Y and 46 Y were approximately 70%and 80%,respectively,histological observation revealed that the hyphae length and hyphae area of avirulent or virelent Pst were both decreased in 9-6Y or 46 Y knockdown plants.Along with that,increased reactive oxygen species and necrosis area were observation in9-6Y and 46 Y knockdown plants when challenged by avirulent Pst.These results showed that silencing of 9-6Y and 46 Y could enhance the resistance of wheat to Pst,which suggested that 9-6Y and 46 Y were negative regulators in wheat disease resistance.Taken together,our results indicated that the effectors Pst＿9302 and Pst＿148 inhibit the wheat immune response by regulating 9-6Y and 46 Y,thereby promoting the pathogenicity of Pst. |
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